Chlorination inhibitors in chlortetracycline-tetracycline fermentations



United States Patent 9 i f N H E AT Joseph 1560B cbbdii a yNiinut, N.Y., and Richard Wil: liamYbun Riverside, Conn., assignors to -American Cyanarnid-Companfl New York, vll.Y., a corporatioui I of Maine a v, .7

NdDrawing. Application November 2, 1956 -Serial No.619,947- v 1 12Cla'ms. (Cl. 195-80) I This rention re t s t6, as assassins 9f te a- Y,- cline and mor part c a y s. con erne w t inhi iips e n ma ive. r hlt in ti n h ertet li i ra c in men a i n whe eb h h yields 1 f et acycline are produced." t y it' a been iswver si t t c g n sms of the genus Streptomyc'es which produce chlor'tetracycline, will also produce tetracycline particularly the chloride ion concentration ot thefe'r'rneritationgmedium is] kept low. This'can'be donefby providing fermentation' media from which chloride "ions are excluded; either by making up the fermentation; medium with chloride-free mmunism by t at n .thsnesiunwitha ent which.

remove or sequester the chloride; ions, thus making them unavailableifor the formation of chlortetracycline.

Unfortunately, sorrieof the most effective components of fermentation media tain suhstantial quantities of chloride ions.

antihioticscon Corn steep"liquorlis'jone 'of the most, eifectivenutrient substances for thefpljpdllbtitjn of thetetracycline antibiotics as; well as many other'antibiotics. Apparently, this natural material contains somethingthathis especiallyUde-i sired by the fermenting microorganism. Highest yields of antibiotic are, therefore, obtained 7 when a portion of corn steep liguor'isjincluded infthe. aqueoussnutrient medium.- 7

Corn steep liquor contains a substantial amount of.

chloride ions; however, anrljif formation of chlortet'racycline is to be kept at reasonably low levels, it has here tofore been considered necessaryitosreduce the-chloride;

content of-thi's material. The same applies to a number of-other naturally occurring nutrientmaterials which are advantageously used in thefermentation process;

"Several means of reducing the chloride ion content of aqueous nutrient solutions for tetracyclineproduction have beenfsuggested. Precipitation'of-the chloride ion as'silver chloride is a very effective means of reducing the chloride'ion content. 'However, this is an expensive process requiring expensive raw means,spciarequi ofsilver. V. s -t Ion exchange resins have also been proposed for the reduction of the chloride ion content of'fermentation media. These substances tend to'remove some salts and organic substances of unidentified composition which are desirable coiriponentsof thevferrnentation media. lfAs a res tilt, low yields of antibiotic may be obtained when usionexchangelresins to remove chlorideionsQ vT The chloride deprivation systems still leave much to be desired, however, because of the capital investment required, the relatively complicated means of removing chloride ions from the fermentation medium, as well as the'fact that the fermentation medium c'ainfin such instances becomposed only of those raw materials from which chloride ions can be easilyremoved or which are naturally lowin chlorides.

"It has also been proposed to shift the equilibrium in a tetracycline chlortetracycline fermentation by the use of bromide which apparently represses 'chlortetracycline fora ice. i qms ss stst' ing nutrient solutions which have been pre-treated with merit, and'iatoxic ga s,hydrogen sulfide, for, therecovery Eatented Feb. 2, 1960 See The present invention depends for its effectiveness in producing high yields of tetracycline in a fermentation medium in which it is not necessary to remove chloride ions from the system. The present invention is based upon the discovery that when a chlorination inhibitor as hereinafter described is added to a chloride-containing fermentation media, the activities of the microorganism are directed from the synthe sis of chlortetracycline to thesynthesis of tetracycline; "The invention is of considerable" practical importance because itobviates the; expense rid disauvamages of removing-'chlorideiions from;

s th'frin ntation' m edium. Thus, inaccor'd'ancewitli the;

S; sayamaensis; S .-'feofaciens,' and stillothers. Thepiih 1 hibitors as hereinafter described? p e h fermentation can be'easily converted to a tetracycline f miita'tion by the addition to "the" nutrient medium '0 relatively small amount of the novel "chlorination i The present-"invent on is not particularly concerned with'lany specific microorganismsexcept to the ex'tentl' that 'it" is" concerned with" those microorganisms J that; pros, du'ce both chlortetracycline and; t etracycline byfermentaei.

tivebiosynthesis'. lnsofa'r'as' is presently known, all:suchi Regardless of this," however, the present invention: is not; predicated' up'on the selection of a particular species oi microorganism, but as, indicated above, is concerned wi the useof certain chlorinationiinhibitors. which directthef 1 act vities:"of v the microorganism from the synthesis ofa chlortetracycline to; the synthesis of I tetracycline; Cons;

sekjue'ntly, the present: invention includesixany microors. ganism which fproduces 'chlbrtetr'acycline andtetracycline; The chlorination :inhibitors. of this invention may The represented by the following: general formulai s s Y,X,\ ..s N,.0j.Z, Y

Whgrfin i i i m 2ft egrqun consisting nd, IOWW lkyl radicals" such a rfi t propyh, prppyl, butyl, en lg x 9 1 ns s n q t ydrogeu, phenyl,' lower allgy l,

v t p O:

s- M P PY' I= Ws 1 sy anct wherein ,Risphenylor. lower allryl; angs -Z g h h vsr c s ii an :isting of S(lower alkyl) scmas mcoomson "ve'nti on' a chloride-containing chlor'tetracyclirnr;

and

examples appearing hereinafter.

-As in the case of the use of bromide-ions in depressing fermentative chlorination the inhibitors of the present invention may'be somewhattoxic to the microorganism, especially when used in high concentrations. Fortunately, verysmall amountsof the inhibitors of this invention depress the formation of chlortetracycline very markedly. As little as one part per. million shows some effect. Ordinarily the preferred range will be from about five parts 1 per million to 100 parts per million, but we may use 500 parts per million or even more if desired. Generally speaking, the more inhibitor that is added, the less chlortetracycline will be produced. On the other hand, as continued amounts of the inhibitor are added to the-toxic effects begin to exert themselves and the total yield of antibiotic is reduced. Hence, no advantage has been observed thus far in going beyond about 500 parts per million and in some cases even'less.

.It is a further advantage of this invention that the chlorination inhibitors make it possible to use a wide variety of strains of S. aureofaciens. That is to say, that with the chloride deprivation systems oftentimes the highest tetracycline-producing strains, that is the chloride scavenging srains, could not be used because they also produced a high level of chlortetracycline. With this invention, however, such strains may be used with facility as the chlorination inhibitors make it possible to easily'shift the equilibrium in favor of tetracycline.

The conditions of the fermentation are generally the same as for the presently known methods of producing tetracycline or chlortetracycline by fermentation. That is, the fermentation medium contains the usual nutrients and mineral substances. Suitable nutrient substances which may provide those necessary substances include starch, dextrose, cane sugar, glucose, molasses, soybean rneaL'peanut meal, yeast, meat extracts, peptone,'ammonium sulfate, urea, corn steep liquor, distillers solubles, fish meal and other conventional substances. The inorganic salts include such things as calcium carbonate, ammonium sulfate, ammonium chloride, and the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation, such as hydrogen ion concentration, temperature, time, rate of aeration, preparation of the inoculurn, sterilization, inoculation and the like are conventional and may be similar to those'for the production of chlortetracycline shown in the U.S. patent to Duggar No. 2,482,055, and for the production of tetracycline shown in the U.S. patent to Minieri et al. No. 2,734,018.

Similarly, the recovery of the tetracycline from the fermentation liquor is conventional and need not be described, as numerous methods of recovering tetracycline from fermentation liquors have been published.

In the example which follows the yields of tetracycline and chlortetracycline are expressed as gammas per milli- EXAMPLE A chlortetracycline. fermentation medium, such as may be used on large scale production, and containing a large amount of chloride ion was made up as follows:

Corn flour"-.. 14.5 grams per liter.

Starch 47 grams per liter. Corn steep liquor 25 grams per liter. CaCO 9 grams per liter. (NH SO 5.6 grams per liter. MnSO (70%) 80 milligrams per liter. CoCl .6H O 5 milligrams per liter. Lard oil 3% by volume. Ammonium chloride' 1.7 grams per liter.

Table Chlor- Tetra- Percent Compound p.p.m. tetracycllne, etrae cline. 'YlmL. cyeline 0 4.900 685 Q l Ethyl-N-benzoyldithioearba- 50 Y 4, 950 850 g mate 4,075 1,175 22.3 V 200 3.025 1,560 .340 58 725 8.5

- 917 12.5 Ditmooxamldhfi 100 4, 400 947 17. 7 8 #338 it? Tetramethylthiurammonosul- 50 5750 v 1, 950 22:; fide 100 4, 750 2, 840 37. 4

200 1. 925 3, 820 66. 4 g 725 8.5 957 12.1 Tetramethylthiuramdlsulfidm. m0 6 0 L225 mo 200 5. 700 1, 750 23. 4 .2 tea 5 24.0 Tmlmmdlsumde 100 4, 600 2, 150 31. s e 208 2, 2,35 47.5

, 5 1 Bis (ethylmethylthluram)di- 50 6, 500 1, 195 sulfide 100 4, 600 1, 19. 9 208 442 21. 7

, 845 Methyl-N,N -dlmethyldithlo- 50 6. 225 775 g carbamate 100 5, 075 815 13. 8 208 gSg 800 16. 4 a 5 925 12. Benzyl N acetyldithlocarba- 50 5. 800 l, 215 17. g mate i 100 5,300 1,500 22.0 208 1, 325 22. 8 25 12.3 Tert-butyl-N-acetyldithiocar- 50 6, 300 1, 245 16. 5

bamate 100 5. 750 1,615 21. 9

20g 2,195 30.3 925 12.3 Carb0xymethyl-N,N-dlmeth' 50 6. 000 970 13.9 yldithiocarbamate 100 5, 450 1, 345 19. 8 20g 1, 385 21. 7 25 12.3 N,N-dlmethyl-p-chlorobenzyl- 50 2, 800 2. 285 44. 9 dlthiocarbamate 100 5, 050 I, 410 21. 8 200 5. 100 1, 225 19. 3 0 6,550 925 12.3 Methylbenzoyldithiocarba- 50 6, 300 1,185 15. B mate 10 5. 800 1,380 19.2 200 3. 850 1, 965 33. 7 0 6,250 860 12.0 Benzyldithiocarbazate-.- g g' 1 25 I500 2:575 36:3 0 6,250 860 12.0 N formyl benzyldtthmcarba- 5 480 5. 840 .92. 4 zate 10 1, 600 4, 870 75. 2 25 4. 525 2, 360 34. 2 0 6.750 835 11.0 N ueetyl benzyldithiocarba- 5 1, 4, 880 80. 9 zate 10 520 5, 100 90. 7 25 280 5, 260 94 Q r 0 6,750 835 11.0 N-benzoyl-henzyldlthrocarba- 5 530 5. 280 90. 8 auto 10 6.120 96 9 25 45 1.890 97.6

unis" wherein X is a member of the group consisting ofhydrogen and lower alkyl radicals; Y is a member of the group consisting of hydrogen, phenyl, amino, lower alkyl,

II R-C wherein R is a member of the group consisting of phenyland lower alkyl, and

wherein R is a member of the group consisting of phenyl and lower alkyl; and Z is a member of the group consisting of S(1ower alkyl),

serif-Q01 which inhibits the formation of chlortetracycline and causes the formation of substantial quantities of tetracycline.

2. A process according to claim 1 in which the chlorination inhibitor is N-acetyl-benzyldithiocarbazate.

3 A process according to claim 1 in which the chlorination inhibitor is N-benzoyl-benzyldithiocarbazate.

4. A process according to claim 1 in which the chlo rination inhibitor is N-formyl-benzyldithiocarbazateQ 5. A process according to claim 1 in which the chlorination inhibitor is N,N-dimethyl-p-chlorobenzyldithiocarbamate. 2

6. A process according to claim 1 in which the chlorination inhibitor is tetramethylthiurammonosulfide. v

7. A process of producing tetracycline by aerobic fermentation of a chloride-containing aqueous fermentation medium with a tetracycline-producing strain of S. aureofacz'ens which comprises the step of adding to said medium from about 5 to 500 parts per million of a chlorination inhibitor of the formula:

wherein X is a member of the group consisting of hydrogen and lower alkyl radicals; Y is a member of the group consisting of hydrogen, phenyl, amino, lower alkyl,

0 II R-G wherein R is a member of the group consisting of phenyl and lower alkyl, and

wherein R is a member of the group consisting of phenyl and lower alkyl; and Z is a member of the group consisting of S(1ower alkyl),

sent-Q01 and References Cited in the file of this patent UNITED STATES PATENTS 2,734,018 Minieri et al Feb. 7, 1956 FOREIGN PATENTS 316,291 1 Switzerland Nov. 15, 1956 OTHER REFERENCES Schwarzenbach et a1., Helvetica Chimica Acta, 29 (1946), pages 364-370. Ibid, Vol. 31, pages 456-459.

Martell et al., Chemistry of the Metal Ohelate Compounds, 1952, Prentice Hall, Inc., Edgewood Cliffs, N.J., pages 6, to 139, 168 to 171 and 471, 506.

Sekizawa, Iour. of Biochemistry, vol. 42, No. 2, March 1955, pages 217-218. 

1. IN A PROCESS OF PRODUCING TETRACYCLINE GY AEROBIC FERMENTATION OF AN AQUEOUS FERMENTATION MEDIUM WITH A TETRACYCLINE -PRODUCING MICROORGANISM OF THE GENUS STREPTOMYCES, THE IMPROVEMENT WHICH COMPRISES ADDING TO SAID MEDIUM A SMALL BUT EFFECTIVE AMOUNT OF A CHLORINATION INHIBITOR OF THE FORMULA: 